Ethanol ingestion produces rapid changes in hepatic mitochondrial metabolism. These changes include a swift increase in alcohol metabolism (SIAM) by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ACDH), a near doubling of respiration, decreased ATP, activated glycolysis, glycogen depletion, inhibition of mitochondrial oxidation of medium and long chain fatty acids, and fatty liver changes (steatosis). The unifying hypothesis of this proposal is that closure of mitochondrial voltage dependent anion channels (VDAC) is a critical event leading to these acute alterations of mitochondrial metabolism after ethanol. VDAC is located in the mitochondrial outer membrane and conducts solutes of molecular weight less than 5 kDa into and out of the mitochondria. Open VDAC is essential for movement of respiratory substrates (e.g., medium and long chain fatty acids, pyruvate), ATP, ADP, Pi and other polar metabolites into and out of mitochondria. We specifically hypothesize that VDAC closure after ethanol blocks these exchanges. After VDAC closure, mitochondria still oxidize membrane permeant respiratory substrates, particularly acetaldehyde and short chain fatty acids. Inhibition of ATP release and the high redox potential of [unreadable] acetaldehyde/acetate then produce mitochondrial hyperpolarization, leading to non-ohmic proton leaks in the hperpolarized inner membrane and respiratory stimulation. Activation of uncoupling proteins and the weak uncoupling effect of acetate may also contribute to increased respiratory flux. Overall, accelerated respiration and selective oxidation of acetaldehyde have the physiological function of removing and detoxifying ethanol. Heretofore, examination of VDAC conductivity in situ has been very difficult. In our preliminary studies, we developed a novel confocal microscopic technique utilizing the distribution of a 3 kDa rhodamine-conjugated dextran in the mitochondria of permeabilized hepatocytes to assess the open/closed status of VDAC. These pilot experiments support the hypothesis that acute ethanol exposure induces VDAC closure. Accordingly,our Specific Aims are 1) to characterize changes of VDAC permeability in rat hepatocytes exposed to ethanol from measurements of respiration and RhoDex retention by permeabilized hepatocytes and isolated mitochondria; and 2). to determine which metabolites of ethanol oxidation (NADH, ROS, acetaldehyde,acetate) are responsible for ethanol-induced VDAC closure or whether closure is a direct effect of ethanol. Overall, this proposal addresses the high risk, high return hypothesis that VDAC closure after ethanol exposure causes mitochondrial dysfunction and promotes steatosis. If our preliminary data supporting this hypothesis can be extended, this new role of VDAC will represent a paradigm shift in our understanding of the effects of alcohol on the liver and the regulation of mitochondrial metabolism. [unreadable] [unreadable] [unreadable]